Grain production with field conditioned pollen

ABSTRACT

Described are methods of grain production with field conditioned pollen. A method of the present invention includes growing one or more designated female plants that include both female and male components, collecting fresh pollen from designated male plants from a different genetic background, and subjecting the fresh pollen to field conditions, which may regulate pollen moisture content. The field conditioning conditions may include a relative humidity ranging from about 50% to about 100%, a temperature ranging from about −10-10° C., and an air pressure ranging from about 15 kPa to about 150 kPa. These conditions may result in pollen having a moisture content of about 40% to about 58%. The one or more designated female plants are intentionally cross-pollinated with the field conditioned pollen and then grown to maturity such that the grain may be harvested therefrom. Also provided is a method of preventing undesirable pollination in grain production.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/803,565, filed on Feb. 27, 2020 and entitled “Cereal Crop PollenField Conditioning Method”. U.S. patent application Ser. No. 16/803,565is a continuation of U.S. patent application Ser. No. 15/486,737, filedon Apr. 13, 2017 and entitled “Pollen Field Conditioning andPreservation Method”. U.S. patent application Ser. No. 15/486,737 is acontinuation-in-part of U.S. Non-Provisional patent application Ser. No.15/192,519, filed on Jun. 24, 2016 and entitled “Grain Production”,which claims priority from U.S. Provisional Application Ser. No.62/184,596 filed Jun. 25, 2015 and entitled SEED PRODUCTION and fromU.S. Provisional Application Ser. No. 62/269,496 filed Dec. 18, 2015 andentitled SEED PRODUCTION and from U.S. Provisional Application Ser. No.62/269,531 filed Dec. 18, 2015 and entitled GRAIN PRODUCTION and fromU.S. Provisional Application Ser. No. 62/269,514 filed Dec. 18, 2015 andentitled GRAIN PRODUCTION and from U.S. Provisional Application No.62/321,914 filed Apr. 13, 2016 and entitled POLLEN CONDITIONING ANDSTORAGE METHOD. The contents of U.S. Non-Provisional patent applicationSer. Nos. 15/192,519; 15/486,737; and 16/803,565 and ProvisionalApplication Ser. Nos. 62/184,596; 62/269,496; 62/269,531; 62/269,514;and 62/321,914 are hereby incorporated in their entireties by reference.

This invention was made with government support under USDA SBIR Phase 1Award No. 2016-33610-25366 titled Development of Rigorous and ReliableMethods to Preserve Maize Pollen awarded by the United States Departmentof Agriculture (USDA). The government has certain rights in theinvention.

FIELD OF THE INVENTION

This invention relates generally to a novel pollen field conditioningand preservation method for increasing the overall viability andfertility of pollen for use in making pollinations conducted with eitherfresh pollen or pollen that has been preserved.

BACKGROUND

The current invention has application to the field of pollen longevityand viability. Pollen longevity varies significantly among species andis significantly influenced by environmental conditions, most notablytemperature and relative humidity. Pollen, which is naturally shed fromthe flowers or flowering structures of angiosperms, is subject to rapidloss of viability once it is shed from the plant. Viability can be lostin minutes to hours depending on species and environmental conditions.Exposure to dry air and high temperature is particularly detrimental topollen viability and longevity once it is shed from the plant. Thus,under natural field conditions, pollen has a limited lifespan duringwhich it remains viable, referred to in this application as the“viability window.” In particular, pollen from the Poaeceae (Gramineae)family of plants, commonly referred to as grasses, is particularlyvulnerable and short-lived (Barnabas & Kovacs (1997) In: PollenBiotechnology For Crop Production And Improvement. (1997). Sawhney, V.K., and K. R. Shivanna (eds). Cambridge University Press. pp. 293-314).This family of plants includes many economically important cereal crops,including maize. Methods to improve pollen viability and extend theduration of its viability are of significant value to the agriculturalindustry.

Specifically, if pollen collected from plants can be stored in a viablestate for a period of time, this pollen may be used to pollinate femaleflowers as desired in a number of advantageous ways. Utilizing storedpollen allows for pollination which is not dependent on active pollenshed, temporal synchrony with pistil (female flower) receptivity, use ofmale sterility, and/or physical isolation from other pollen sources.Currently, many species rely on self-pollination or cross pollination byneighboring plants to produce fertile seed or grain. Typically in theagricultural hybrid seed industry, mechanical, physical, and/or geneticinterventions are required to ensure female plants are cross pollinated,and not self-pollinated, so that pollen of a specific geneticconstitution is employed to produce hybrid seed. Such measures, forexample, are used routinely to produce hybrid maize and rice seed. Insome crops, however, even these measures are not effective to ensurecost-effective cross pollination by a specific desired pollen source.Currently, it is not possible, or is very difficult to, produce thesecrops commercially as hybrids. Examples of these crops include, but arenot limited to, wheat and soy.

Many attempts have been made to preserve pollen and extend its viabilityfor pollinations beyond the time the pollen would remain viable if leftexposed to uncontrolled ambient conditions. Among the grasses, studieswith maize are exemplary of the progress made in pollen preservation.Many types of treatments have been tested for maintaining or extendingmaize pollen viability and/or fertility. Among them, the favorability oftreating and/or storing maize pollen at high humidity and/or coldtemperature has been reported by many.

Among the earliest accounts of maize pollen preservation (Andronescu,Demetrius I., The physiology of the pollen of Zea mays with specialregard to vitality. Thesis for degree of Ph.D. University of Illinois.1915), it was reported that in the absence of controlled environmentalstorage conditions, pollen died in two to four hours. By raising therelative humidity of the storage environment, the pollen's viability wasmaintained for 48 hours. Moreover, storage at low temperature (e.g.,8-14° C.) had a stimulative effect upon the viability of the pollen.

Even when relative humidity is not controlled during storage, maizepollen held at low temperature (e.g., 2-7° C. for 3-120 hours) can morethan double its in vitro germinability compared to initial, pre-storagevitality or compared to storage at 35° C. (Pfahler, P. L. and Linskens,H. F., (1973) Planta, 111(3), pp.253-259; Frova, C. B. and Feder, W. A.,(1979) Ann Bot, 43(1), pp.75-'79). When high humidity (90% RH) and lowtemperature (4° C.) during storage are combined for pollen treatment,germination of maize pollen on artificial media remains good, to fair,for eight days (Sartoris, G. B., (1942) Am J Bot, pp.395-400). Storageof maize pollen under the same conditions for eight days also allows thepollen to remain fertile, albeit at a reduced level, and capable offorming kernels on ears following pollination (Jones, M. D. and Newell,L. C., (1948) J Amer Soc Agron 40:195-204).

Field conditioning maize pollen at high humidity and low temperaturecommonly help revive pollen of low viability and/or extend itslongevity, whereby at least limited seed formation occurs followingpollination of ears. But the stimulative effect of low temperaturestorage on fertility is not always observed (Walden, D. B., (1967) CropScience, 7(5), pp.441-444) and if the pollen becomes dehydrated toexcessive levels, pollen tube formation on artificial media and silkscan be markedly reduced (Hoekstra, FA. (1986) In: Membranes, Metabolismand Dry Organisms. (Ed., AC Leopold), pp. 102-122, Comstock PublishingAssociates, Ithaca, N.Y.; Barnabas, B. and Fridvalszky, L., (1984) ActaBot Hung 30:329-332).

Although high humidity and low temperature slow the temporal decay ofviability during storage of Gramineae pollen, optimizing theseenvironmental conditions for preservation only postpones the completeloss of viability and fertility. Methods in addition to regulatinghumidity and temperature are needed to further enhance the longevity ofstored pollen so that it can be used in commercial practice ofsupplemental pollination for improved seed and grain production.

In some cases, it may be desirable to treat pollen so that it isdehydrated to various degrees. Dehydration can be achieved by vacuumdrying or exposing pollen to a relative humidity and temperature (i.e.,vapor pressure deficit) that causes water to diffuse out of the pollen.Vapor pressure deficits favorable for pollen drying can be produced in anumber of ways, such as with desiccants, mechanical equipment designedto control temperature and relative humidity in an enclosed chamber andwith saturated salt solutions held in a closed space (Jackson, M. A. andPayne, A. R. (2007) Biocontrol Sci Techn, 17(7), pp.709-'719),Greenspan, L., (1977) J Res Nat Bur Stand, 81(1), pp.89-96)

In an effort to dehydrate and preserve sugarcane pollen, the pollen wasstored at low temperature under vacuum with a small amount of CaCl₂desiccant present (Sartoris, G. B. (1942) Am J Bot, pp.395-400). Thepollen remained dry throughout storage, as desired, but use of lowpressure was not as favorable as storage at normal atmospheric pressure.The behavior of corn pollen was very similar to that of sugarcane. Moredirect attempts at dehydration have incubated pollen in conditions ofestablished or recorded relative humidity and temperature. Theseexamples show that maize pollen can be dehydrated to very low levels(e.g., 7-10% pollen water content) and still possess an ability, albeitreduced, to effect seed formation following pollination of ears(Barnabas, B., et al. (1988) Euphytica, 39(3), pp.221-225; U.S. Pat. No.5,596,838).

Dehydration of pollen is commonly performed ahead of freezing forstorage and preservation at very low temperatures. As practiced withmaize, fresh pollen is dehydrated at room temperature in a vacuumchamber, humidity incubator, or simply with air-drying or mild heat(U.S. Pat. No. 5,596,838; Barnabas, B. and Rajki, E. (1981). Ann Bot,48(6), pp.861-864; Connor, K. F. and Towill, L. E. (1993) Euphytica,68(1), pp.77-84). Upon thawing after short or long term storage,cryopreserved pollen can be viable and fertile, but fertility is notalways exhibited and some members of the Gramineae family, such asmaize, sorghum, oat and wheat, can be difficult to cryopreserve(Collins, F. C., et al. (1973) Crop Sci, 13(4), pp.493-494). Oneexplanation offered for this recalcitrance is excess drying or aging ofthe pollen (Collins, F. C., et al. (1973) Crop Sci, 13(4), pp.493-494).It is evident that pollen quality can be affected by prevailingenvironmental conditions during floral development, pollen maturation,and anthesis (Shivanna, K. R., et al. (1991) Theor Appl Genet 81(1),pp.38-42; Schoper, J. B., et al. (1987) Crop Sci, 27(1), pp.27-31;Herrero, M. P. and Johnson, R. R. (1980) Crop Sci, 20(6), pp.796-800).Pollen stressed in these ways could exhibit a reduced propensity towithstand the rigors of dehydration and freezing for cryopreservation. Aneed exists to overcome this problem and make cryopreservation ofGramineae pollen more attainable and routine so this form of pollenpreservation can be implemented in a predictable way on a commercialscale.

Desiccation is known to have a direct impact on pollen viability.Barnabas (1985) Ann Bot 55:201-204 and Fonseca and Westgate (2005) FieldCrops Research 94: 114-125 demonstrated that freshly harvested maizepollen could survive a reduction in original water content ofapproximately 50%, but few pollen grains demonstrated viability or acapacity for normal pollen tube formation with an additional water lossbeyond that level. Early work by Barnabas and Rajki ((1976), Euphytica25: 747-752) demonstrated that pollen with reduced water content wouldretain viability when cryogenically stored at −196° C. Subsequent work(Barnabas & Rajki (1981) Ann Bot 48:861-864) demonstrated that suchpartially-desiccated maize pollen grains stored at −76° C. or −196° C.also could effect fertilization of receptive female flowers. Othermethods of storing pollen for varying periods of time are known in theart, including freeze-drying, vacuum-drying, and storage in organicnon-polar solvents. Limitations in the scalability of these pollenpreservation techniques combined with the complex, non-portableequipment requirements render these techniques impractical for use withlarge volumes of pollen required for field-scale applications.

U.S. Pat. No. 5,596,838 from Greaves, et al., discloses a method ofstoring pollen that involves a reduction in moisture level by exposingpollen to reduced atmospheric pressures prior to storage. This techniqueprepared small quantities of pollen, such as from a single maize plant,for subsequent storage under sub-zero conditions. The Greaves et al.method has drawbacks. For example, the methodology and mechanical systemrequirements lack the capacity to produce stored pollen in quantitieslarge enough to enable commercial seed production or grain productionapplications. These requirements effectively negate any opportunity toadvance the technology beyond research level investigations. Forexample, the ability to create a vacuum chamber large enough forproduction-level field pollination preservations would require a verylarge vacuum chamber capable of rapidly changing pressure levels.Production-level parent increase fields are typically an acre or more,while hybrid production fields are typically 10 acres or more in size.Such fields require a considerable amount of pollen and thus a largevacuum chamber would be needed. A chamber of the Greaves, et al.specifications would require the ability to pump down to a pressure of 5Torr (0.67 kPa) or less, with the added ability to rapidly up cycle anddown cycle this level of pressure. As the physical volume of the sampleincreases, the ability to generate and cycle at 5 Torr (0.67 kPa)efficiently begins to go beyond what mechanical pumps can generate. Inaddition, storage of pollen in organic solvents creates hazardouschemical requirements.

The availability of preserved, viable pollen would overcome many of theproduction challenges faced by the hybrid seed industry. As provided infurther detail in Applicant's U.S. patent application Ser. No.15/192,485, the entire contents of which are hereby incorporated byreference, with respect to hybrid seed, the availability of storedpollen for delivery to female flowers can eliminate many standard,costly practices of seed production including, but not limited to,planting male plants in proximity to female plants to enablehybridization, isolation of female plants from undesired pollen sources,and use of genetic or mechanical male sterility of the female plants.These practices dramatically increase field space and resourcesdedicated to female plants which produce seed or grain. Reducing oreliminating any one of these practices would reduce the cost of hybridseed production. Moreover, stored pollen can be applied at any time.When pollen shed from male plants and pollen receptivity of femaleplants fail to coincide as planned (due to management, environment, orgenetic variation), application of preserved, viable pollen can ensurepollination of female plants at the optimal time. Pollination byundesired external (adventitious) sources of pollen or undesiredself-pollination of female plants also can be reduced or eliminated byapplying stored pollen of a desired type at the appropriate time. Today,the genetics of a particular hybrid seed is determined at the beginningof a growing season by the genotype of the pollen-donating male plantsand pollen-receiving female plants planted together in the field. Usingthe embodiments of the present disclosure, however, a hybrid seedproducer responding to changing market opportunities can decide at thetime of pollination to use a different male pollen (i.e., geneticsource) for pollination to produce more valuable hybrid seed. Inaddition, stored pollen can be used to deliver unique genetic traits orgenes that enhance seed quality characteristics to highly productivefemale inbreds. For example, traits for resistance to select insectpests which are present might be delivered. Importantly, the embodimentsof the present disclosure also ensure a high level of genetic purity inthe hybrid seed. As such, methods of improving pollen viability formultiple crop species and extending the duration of its viability are ofsignificant value to the agricultural industry.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: This figure demonstrates the effect of ambient laboratoryconditions of 21° C. and 30% relative humidity on the viability offreshly-shed pollen over time.

FIG. 2: This figure demonstrates the variability in the pollen moisturecontent of freshly-collected pollen from the field.

FIG. 3: This figure shows the effect of field conditioning pollen bycomparing the in vitro germination rates of pollen immediately aftercollection and again two hours later following a period of fieldconditioning.

FIG. 4: This figure demonstrates the average pollen germinationpercentages of four maize genotypes following storage of the pollen inhigh humidity conditions for one hour at either 4° C. or 22° C.

FIG. 5: This figure shows the effect of field conditioning onrevitalization of pollen viability across multiple genotypes. For eachgenotype, the effects of revitalization in response to high humidity andthe advantage obtained by field conditioning at low temperature areshown.

FIG. 6: This figure shows the change in pollen moisture content indifferent genotypes based on the time of day that the pollen collectiontook place.

FIG. 7: This figure demonstrates the effect on pollen moisture contentof storing freshly-collected pollen for 6 days at two different pressurelevels while maintaining a high humidity level.

FIG. 8: This figure shows the change in pollen germination percentagefollowing the storage of freshly-collected pollen for 6 days at twodifferent pressure levels, both at a high humidity level.

FIG. 9: This figure shows the change in pollen moisture content acrosssix different genetic backgrounds at time zero, and after six days ofstorage at different air pressure values.

FIG. 10: This figure shows the variation in pollen germinationpercentages across six different genetic backgrounds at time zero, after1 day of field conditioning at ambient air pressure, and after six daysof storage at different air pressure values.

FIG. 11: This figure demonstrates the viability of pollen from eightdifferent genetic backgrounds after field conditioning with highhumidity and low temperature at time zero, and then after 3, 5 and 8days of storage at air pressure values of either 101.3 kPa or 67.4 kPa.

FIG. 12: This figure shows the dehydrating effect of nitrogen gas onpollen moisture content during a four-hour period of exposure and howdehydrating pollen to varying degrees affects its viability when storedfor 17 days.

FIGS. 13A and 13B: These figures show the pollen moisture content ofpollen stored at varying relative humidity levels over an eleven-dayperiod.

FIGS. 14A and 14B: These figures show the viability of maize pollenstored at varying relative humidity levels over an eleven-day period.

FIG. 15: This figure shows an image of rice pollen stored at 4° C. and100% relative humidity germinating in culture after 20 hours ofpreservation.

FIG. 16: An image of rice pollen stored at 4° C. without relativehumidity control germinating in culture after 20 hours of preservation.Overall germination for this treatment was measured at 1%.

FIGS. 17A, 17B, and 17C show the results of pollinations conducted withpreserved pollen following storage periods of 4 hours to 38 days asindicated in example 10 and Table 3.

FIG. 18: This figure shows the final germination media results fromrapidly frozen and rapidly thawed maize pollen. Overall germination wasscored at less than 0.5%.

SUMMARY OF THE INVENTION

Provided is a method of grain production comprising growing a pluralityof designated female plants that include both female and malecomponents, collecting fresh pollen from designated male plants from adifferent genetic background, subjecting the pollen to fieldconditioning conditions including a relative humidity ranging from about50% to about 100%, a temperature ranging from about −10° C. to about 10°C., and air pressure ranging from about 15kPa to about 150kPa, resultingin field conditioned pollen having a pollen moisture content of about40% to about 58%. The designated female plants are intentionallycross-pollinated, on one or more days, with the field conditionedpollen. The designated female plants are grown to maturity, and theirgrain is harvested. The female components of the designated femaleplants are not covered to prevent undesirable pollination.

In some embodiments, the field conditioned pollen may be preserved inpollen preservation conditions including a temperature ranging fromabout −10° C. to about 10° C. and an air pressure that is capable ofadjustment, resulting in a pollen moisture content of about 15% to about35%. The temperature and relative humidity may be adjustable to maintainthe pollen moisture content at about 15% to about 35%.

Methods of the invention may result in one or more of increased grainyield and modified grain characteristics. Modified grain characteristicsmay include one or more of grain size, grain content, and graincomposition. In some cases, the designated male plant pollen mayinfluence the characteristics of the grain harvested from the femaleplant, such as grain oil content, starch content, protein content, oilcomposition, starch composition, and/or protein composition.

In the case of fresh pollen, the pollen may have been harvested from oneor more of a field, growth chamber, greenhouse, glasshouse, shade house,hoop house, vertical farming facility, or a hydroponic facility. Thedesignated male plant pollen may be obtained from sources optimal forapplication based on environmental conditions, including but not limitedto abiotic conditions such as drought, humidity, temperature, nitrogenavailability, and nutrient availability. In other embodiments, theenvironmental conditions may be biotic conditions, including but notlimited to insect pest pressure and disease pressure. Furthermore, thedesignated male plant pollen may be obtained from sources optimal forapplication based on plant performance data. The designated male plantpollen may be obtained from a single genetic source or multiple geneticsources. Moreover, the designated female plant may be any one or more ofa corn plant, a soybean plant, a wheat plant, a rice plant, a sunflowerplant, a canola plant, a sorghum plant, a barley plant, or a pearlmillet plant.

Intentional application of designated male plant pollen may be by one ormore of mechanical means, pneumatic means, positive pressure means,negative pressure means, or combinations thereof. In some embodiments,the designated male plant pollen may be applied on more than oneoccasion. Application may occur at the time when the female plant firstbecomes receptive to pollen.

In another embodiment of the invention, a method of preventingundesirable pollination in grain production is provided. The methodincludes growing a designated female plant that includes femalecomponents that receive designated male plant pollen. Fresh pollen iscollected from designated male plants from a different geneticbackground. The fresh pollen is subjected to field conditioningconditions including a relative humidity ranging from about 50% to about100%, a temperature ranging from about −10° C. to about 10° C., and airpressure ranging from about 15kPa to about 150kPa, resulting in fieldconditioned pollen having a pollen moisture content of about 40% toabout 58%. The designated female plants are intentionally pollinated onone or more days with the designated male plant pollen prior to thedesignated female plant being subjected to undesirable pollen on the oneor more days. The designated female plants are not isolated to preventundesirable pollination.

In some embodiments, the field conditioned pollen may be preserved inpollen preservation conditions including a temperature ranging fromabout −10° C. to about 10° C. and an air pressure that is capable ofadjustment, resulting in a pollen moisture content of about 15% to about35%. The temperature and relative humidity may be adjustable to maintainthe pollen moisture content at about 15% to about 35%.

In some embodiments, intentional pollination may occur during themorning. The method may result in a level of cross-pollination that ismodified compared to natural pollination.

DETAILED DESCRIPTION

The following is a detailed description of an embodiment of technologyand methods enabling improved and extended viability of collectedpollen, including techniques of field-conditioning pollen and subjectingit to specialized preservation techniques. The pollen may be collectedfrom actively shedding plants or, alternately, the pollen to be fieldconditioned may have been previously collected and stored according tonumerous methods known in the art that maintain pollen viability over aperiod of time. Such methods include, for example, freezing,freeze-drying, storing in liquid nitrogen, etc.

In order to field condition pollen for improved and extended viability,the pollen is maintained in, or transferred to, a storage chamber thatpermits the modification and/or maintenance of changes to one or more ofrelative humidity, temperature, atmospheric pressure, and gaseouscomponent concentrations of the atmosphere present in the chamber. Forthe purposes of this invention, the term “chamber” is used to mean anenclosure suitable for containing and storing pollen. A chamber can varyin size and material of construction. The chamber may be of any sizethat is suitable for containing a quantity of pollen, and may be anykind of container, vessel, enclosure, or space in which pollen is beingstored, wherein the space serves as a chamber for the storage of largequantities of pollen. In all cases, the chamber must be one in which theenvironmental conditions can be regulated for selected parameters suchas, but not limited to, temperature, relative humidity, composition ofgases, and pressure. The chamber may be equipped with a vent or othermeans to allow pressure to escape from the system and to allow moistureremoved from the pollen to be removed from the chamber.

For the purposes of this disclosure, the term “viable” or “viability” isused to describe pollen that is able to germinate and grow a pollen tubeto at least a length twice the diameter of the pollen grain. Inaddition, pollen can be judged viable by demonstration that the cellularnature of the material remains integral and is judged to maintainintactness such that normal cellular processes of metabolism andintracellular functioning is possible. The viability of pollen can beassessed in numerous ways, including, but not limited to, assessment ofpollen tube growth on artificial media or excised stigmas or styles,assessment of cellular intactness by vital staining of numerous sorts,absence of electrolyte (e.g., potassium) leakage, and impedance flowcytometry. Viable pollen can successfully germinate and commonlypossesses the vigor necessary to promote fertilization and initiation ofseed development. Not all viable pollen is also fertile pollen. In somecases, even when a pollen grain is viable and commences with pollen tubegrowth, it may lack the vigor necessary to reach the ovule and promotefertilization. Non-viable pollen grains cannot successfully germinate.Viability can refer to a single pollen grain or a population of pollengrains. When a percentage value is used to describe pollen viability,the value is typically being applied to a population of pollen.

Another term that can be used to describe the ability of pollen togerminate and form the pollen tube is “germinability.” Thus, pollen withgood viability is pollen that is desirable for use with the methods inthe present disclosure. The “viability window” refers to the limitedlifespan during which pollen remains viable.

For the purposes of this disclosure, the term “fertile” or “fertility”is used to describe the ability of pollen to deliver the sperm nuclei tothe ovule and thereby effect double fertilization. In flowering plants,the term “double fertilization” refers to one sperm nucleus fusing withthe polar nuclei to produce the endosperm tissues, and the other spermnucleus fusing with the egg nucleus to produce the embryo.

For the purposes of this disclosure, “loss of viability” and “loss offertility” are terms used to describe pollen. These terms mean,respectively, that the viability and fertility of the pollen has fallento a level below that required for successful initiation of seeddevelopment. The level of viability and fertility required forsuccessful pollinations is typically defined to be an average of 4grains of fresh pollen per ovule, or 4 to 8 grains of preserved pollenwhich has been preserved according to the methods of the presentdisclosure.

For the purposes of this disclosure, the term “longevity” is used todescribe the length of time that pollen remains both viable and fertile.

For the purposes of this disclosure, the term “pollen fieldconditioning” or “field conditioning” is used to describe the processdisclosed in this application of treating freshly-collected pollen inthe field to maintain or improve its viability, allowing for moresuccessful pollen storage. Storage success is measured by the percentageof a population of pollen that remains both viable and fertilethroughout a period of storage. Field conditioning typically takes placein the field where the pollen is being collected, such that the fieldconditioning is conducted prior to transporting the pollen to alaboratory or other location. It is an immediate process that isconducted upon the gathering of the pollen. Field conditioning may alsotake place in other locations where plants are grown and where pollencollection takes place, such as a growth chamber, a greenhouse, aglasshouse, a shade house, a hoop house, a vertical farming facility, ahydroponic facility, or any growing facility providing cultured tassels,as described below. Field conditioning includes any intentionalregulation of environmental conditions (e.g., relative humidity,temperature, gaseous composition, pressure, light, etc.) of a confinedspace in which freshly-collected pollen is held immediately followingits collection and prior to transportation to any other location.Periods of field conditioning can be brief, on the order of minutes tohours, or extended up to several days. The term “revive” or “revival” isused to describe the nature of the pollen during the field conditioningprocess. For example, to “revive” pollen during the field conditioningprocess may include improving one or more of the viability,germinability, or fertility of the pollen.

For the purposes of this disclosure, the term “storage” means any periodof pollen containment with the intent of using the pollen at a latertime or date. The term “preservation” means any storage of pollen thatresults in a level of viability, fertility, or both, which is differentthan the level of viability, fertility or both, which would occur if thepollen were held in unregulated ambient environmental conditions. Pollenmay, optionally, be field-conditioned prior to storage or preservation.

For the purposes of this disclosure, the term “female plant” is used tomean a plant that is being used as the recipient of the pollen, andwhich has receptive flowers that are being fertilized. In the case ofmaize, and many other species, the plant is monoecious and contains maleand female inflorescences on a single plant. In the practice ofbreeding, pollination, cross-pollination, and hybridization, some plantsact as the “male plant” from which pollen is collected for use inpollinations, and some plants act as the “female plant” being therecipient of the pollen. In the case of self-pollinations, a singleplant is acting as both the “male plant” and the “female plant” becausethe female flowers are fertilized by pollen from its own male flowers.

For the purposes of this disclosure, the term “fresh” when applied topollen means pollen released from the anthers of a flower which, in itsnatural pattern of organ growth and development, releases pollen upondehiscence in response to promotive environmental conditions.

The collection of fresh pollen may be conducted in ways commonly knownin the art. For example, pollen may be collected from freshly sheddingflowers or male flower structures produced in any variety of manners. Inthe case of maize, for example, pollen is collected fromfreshly-shedding male flowers borne on tassels, which may be attached,or detached, from the plant. The pollen may be collected from plantsgrown in any environment suitable for plant growth. Such environmentsinclude, but are not limited to, a field, a growth chamber, agreenhouse, a glasshouse, a shade house, a hoop house, a verticalfarming facility or a hydroponic facility. Alternatively, pollen may becollected directly from anthers by crushing or grinding the anthers,thereby releasing the pollen and allowing for its collection. Inaddition, pollen may be collected from a tassel culture facility(Pareddy D R, Greyson R I, Walden D B (1989) Production of normal,germinable and viable pollen from in vitro-cultured maize tassels. TheorAppl Genet 77:521-526.). Cultured tassels may be mature tassels thathave been removed from plants in any type of growing facility orenvironment, including a field or other type of growing facilities, andplaced into water in a controlled environment to collect pollen orcultured tassels may be tissue that has been harvested from floweringstructures at immature stages and then cultured to develop into a fullymature flowering structure or tassel.

Field conditioning and storage of pollen may be achieved throughplacement of the material into a chamber where environmental conditionsare regulated. For example, a refrigeration chamber with controlledtemperature and the ability to control relative humidity could be usedto field condition and/or preserve the collected pollen. The unit wouldalso have an inlet for flushing various gases or mixtures of gases, suchas nitrogen, carbon dioxide, and/or oxygen, into the chamber. Likewise,a room suitable for storing larger volumes of pollen, which is suppliedby a mechanical form of humidification (sonic, ionized, etc.),dehumidification, is temperature controlled, and permits sampling,monitoring and balancing of gases or gaseous mixture supply in theambient air, is a chamber.

Pollen intended for field conditioning and subsequent preservation andstorage is placed into an environment with high relative humidity, asindicated in various examples, including but not limited to example 4which assesses the effect of relative humidity on the pollen. Therelative humidity may be, for example, any value ranging from about 75%humidity to about 100% humidity, including about 75%, about 76%, about77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%,about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%,about 97%, about 98%, about 99%, and about 100% humidity. At the sametime, the environment must have a relatively low temperature, asprovided in examples including, but not limited to Example 4. Arelatively low temperature may be, for example, any value ranging fromabout −10° C. to about 10° C., including about −10° C., about −9° C.,about −8° C., about −7° C., about −6° C., about −5° C., about −4° C.,about −3° C., about −2° C., about −1° C., about 0° C., about 1° C.,about 2° C., about 3° C., about 4° C., about 5° C., about 6° C., about7° C., about 8° C., about 9° C. and about 10° C. Field conditioning isconducted at an air pressure ranging from about 15 kPa to about 150kPa,including about 15 kPa, about 20 kPa, about 25 kPa, about 30 kPa, about35 kPa, about 40 kPa, about 45 kPa, about 50 kPa, about 55 kPa, about 60kPa, about 65 kPa, about 70 kPa, about 75 kPa, about 80 kPa, about 85kPa, about 90 kPa, about 95 kPa, about 100 kPa, about 105 kPa, about 110kPa, about 115 kPa, about 120 kPa, about 125 kPa, about 130 kPa, about135 kPa, about 140 kPa, about 145 kPa, or about 150 kPa. The fieldconditioning environment may have a flow of one or more continuouslyrefreshed, selected gases, wherein the gas displaces oxygen. Examples ofsuch a field conditioning environment can be found in Example 11 andExample 12. It is anticipated that many gases may be used for thispurpose, including but not limited to inert gases. In some embodiments,gases including nitrogen, carbon dioxide, or combinations thereof may beused. In one preferred embodiment, nitrogen gas may be used. Moreover,the concentration of nitrogen (N₂ gas) present in the chamber may bemoderated from about atmospheric percentage to about 100%, includingabout 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%,about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about97%, about 98%, about 99%, and about 100% nitrogen. The concentration ofoxygen (02 gas) present in the chamber may be moderated from about theprevailing atmospheric percentage to about 0%, including about 21%,about 20%, about 19%, about 18%, about 17%, about 16%, about 15%, about14%, about 13%, about 12%, about 11%, about 10%, about 9%, about 8%,about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, about 1%,and about 0% oxygen.

When the pollen is in this environment with high relative humidity andrelatively low temperature, it is being field conditioned. The period offield conditioning will enhance pollen health and viability. After fieldconditioning, the pollen may be subjected to further preservationtechniques or it may be stored.

In order to achieve a known relative humidity level, a variety of meansknown in the art may be employed. These include, but are not limited to,using an apparatus such as a dew-point generator, an atomizer, amixed-flow generator, a sonicator, or other apparatus designed toincrease relative humidity. It can also be achieved using a two-pressureprocess, a two-temperature process, or a saturated salt solution. Thetwo-pressure humidity generation process involves saturating air ornitrogen with water vapor at a known temperature and pressure. Thesaturated high-pressure air flows from the saturator, through a pressurereducing valve, where the air is isothermally reduced to test pressureat test temperature. Likewise, the two-temperature method circulates anair stream through a precise temperature controlled saturator (waterspray or bubble column). The air becomes saturated at the temperature ofthe water. When leaving the saturator, the air travels through a mistelimination device to insure liquid water does not go beyond thesaturator. The air is then reheated to the desired dry bulb temperature.The temperature of the saturator would equal the dew point temperature.RH is calculated from the dew point and dry bulb temperatures (twotemperature method).

As shown in Example 1, freshly-collected maize pollen from eight diversegenetic backgrounds shows significant variation in viability, even whensampled on a single day under the same conditions. Pollen originatingfrom diverse genetic backgrounds shows significant differences inability to tolerate heat stress and vapor pressure deficits. Subsequentexperimentation on pollen collected from two different geneticbackgrounds (see Example 2) showed that freshly-shed pollen rapidlydeclines in its ability to germinate successfully when exposed toambient laboratory conditions.

The loss of moisture impacts the viability of pollen, with greatermoisture loss resulting in greater viability loss, and eventually thedeath of the pollen grain. The rate of moisture loss is dependent upon anumber of factors, including, but not limited to, ambient temperature,relative humidity, and wind conditions. Pollen death occurs most quicklyin hot, dry conditions, such as those conditions expected during adrought. Placing pollen which is losing moisture content into anenvironment which has high relative humidity and relatively lowtemperature allows the pollen to recover its moisture content almost toits pre-shed level, however, it does not completely stop the pollen frommetabolic activity, which consumes vital resources. This is demonstratedin Example 3, which demonstrates improvement in pollen germination ratesfollowing field conditioning at high humidity and low temperature.Further experimentation demonstrated that field conditioning pollen athigh humidity elevated low viability within one hour, but additionalbenefit was gained from field conditioning at low temperature.

The rate of moisture removal from pollen has a direct impact onpreservation success. The disclosed method allows removal of moisture atrates which exceed what a vacuum can accomplish, which was thepreviously known method for moisture removal from pollen. The disclosednovel process also permits alteration of the rate of drying. Forexample, moisture may be removed at a rapid rate to start with, and thenthe relative humidity levels can be altered in order to reach the targetof 15-30% pollen moisture content at a more gradual rate. Experimentaldata have shown that drying below 15% pollen moisture content results inmuch lower viability of the preserved pollen.

The present disclosure demonstrates that pollen viability can bepreserved, and even enhanced, as a result of field conditioningfollowing collection. While it was previously known thatfreshly-collected pollen of grass species could be stored, it has notpreviously been demonstrated that post-collection field conditioning ofsuch pollen using both temperature and humidity can, on a large-scalebasis, improve pollen viability and enhance storage. A large-scale basisis defined as quantities of pollen measuring about 5 grams or more,including quantities measured as kilograms of pollen. As demonstrated inExample 4, pollen under vapor pressure deficit stress collected from arange of maize inbreds showed significant improvement in percentgermination after being subjected to a brief field conditioningtreatment. The field conditioning treatment served to “rescue” stressed,dehydrated pollen grains thereby reversing their decline in viabilityand improving their capacity for germination. As disclosed in Example 4,the field conditioning of stressed pollen grains with 100% relativehumidity improved germination by 21%, on average. Adding cold treatmentat 4° C. produced a synergistic effect that increased germination by33%. The field conditioning effect was detectable within one hour. Hot,dry conditions, which are typical during peak pollen shed in the field,cause pollen to lose moisture content and expire more rapidly. Thecombined treatment of high relative humidity and low temperature slowspollen metabolism while maintaining moisture content, thereby extendingits viability.

Once the method of field conditioning pollen had been established,further experimentation was done to determine optimal conditions for thestorage of the field conditioned pollen.

Pollen that has been subjected to the field conditioning process cansubsequently be subjected to further preservation techniques or can bestored. If desired, the pollen can be subjected to the fieldconditioning step and then used in pollinations.

Pollen can be subjected to additional preservation steps in order toincrease its ability to retain viability and fertility over storageperiods. It is beneficial to field condition the pollen as previouslydescribed prior to such preservation techniques or storage, in order tomaximize pollen health and viability prior to any storage. Thepreparation of pollen for storage requires a careful process includingan environment with known relative humidity levels, as well as specifictemperature and atmospheric pressure conditions. In addition, thepresence of useful gases will assist in maximizing storage success.

It is initially necessary to remove some moisture from the pollen inorder to maximize the pollen's viability and fertility, as demonstratedin Example 8, and is particularly critical prior to long-term cryogenicstorage. Establishing an environment with a known relative humidityserves to gradually remove moisture from the pollen, thereby acting as adrying agent. Ideally, the relative humidity in the drying environmentshould be any value ranging from about 0% to about 30% relative humidityincluding about 0%, about 1%, about 2%, about 3%, about 4%, about 5%,about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%,about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%about 26%, about 27%, about 28%, about 29%, and about 30% humidity. Thelower the relative humidity level threshold, the faster moisture isremoved from the pollen. The moisture content of fully hydrated pollenis typically about 60%. A target moisture content of the pollen prior tocryogenic storage is a value in the range of about 15% to about 35%,including about 35%, about 34%, about 33%, about 32%, about 32%, about30%, about 29%, about 28%, about 27%, about 26%, about 25%, about 24%,about 23%, about 22%, about 21%, about 20%, about 19%, about 18%, about17%, about 16% and about 15% moisture content. The relative humidity canbe adjusted over time to allow the rate of drying to increase ordecrease. In the field, pollen which falls below 30% moisture contenthas lost viability completely for in vitro germination (Fonseca andWestgate (2005) Field Crops Research 94: 114-125). By contrast, in thepresent invention, pollen fertility and viability are maintained whenpollen moisture content is reduced under cold conditions in anenvironment that achieves target pollen moisture content. The criticalfactor is a very controlled drying process, which cannot be achieved inthe field.

The process of reducing the pollen moisture content may be conducted atdifferent temperatures ranging from −10 to 25° C.

The addition of targeted pressure treatment to the pollen showed thatpollen viability during storage with high humidity and low temperaturemay be improved if stored at specific pressures, which in someembodiments is below atmospheric pressures. This concept is demonstratedby Examples 5, 6, and 7. Specifically, in some embodiments of theinvention, introduction of a vacuum serves to reduce and/or preventoxidative stress on the pollen and, therefore, increase viability.Examples 5-7 below show that viability during storage with high humidityand low temperature is sometimes improved if the pollen is furtherstored below atmospheric pressure. As those skilled in the art willunderstand, the optimal pressure (negative or positive) may varydepending on the species of pollen and the genetic background of thespecific pollen within a species. Moreover, in some embodiments a vacuummay be optimal, whereas in other embodiments pressures at or aboveatmospheric pressure may be optimal. Experimentation based on theexamples provided herein below will allow for optimization of thepressure to maximize the preservation of the pollen's viability.

In maize, for example, storage at about 15 kPa-150 kPa is optimal, asshown in Example 6. Moreover, storage at reduced pressures of about 67kPa-94 kPa may be most optimal, including pressures about 67 kPa, 68kPa, 69 kPa, 70 kPa, 71 kPa, 72 kPa, 73 kPa, 74 kPa, 75 kPa, 76 kPa, 77kPa, 78 kPa, 79 kPa, 80 kPa, 81 kPa, 82 kPa, 83 kPa, 84 kPa, 85 kPa, 86kPa, 87 kPa, 88 kPa, 89 kPa, 90 kPa, 91 kPa, 92 kPa, 93 kPa, and 94 kPa.In some experiments, including Example 6 below, a reduced pressurearound 84.3 kPa may provide for pollen viability which is nearly doubledcompared to storage at atmospheric pressure. Accordingly, storage inreduced pressure, combined with increased humidity and low temperatureimproves pollen viability. Moreover, storage under reduced pressure maymaintain increased viability for all periods of preservation and storageand allow pollen to be held for extended periods for later use. Ofcourse, the optimal pressure conditions and improvements to viabilitymay differ across plant species and within each species across geneticbackgrounds.

Furthermore, in some embodiments of the invention, adjustment of theinitial PMC may provide advantages, as demonstrated below in Example 8.In some embodiments, dehydration of the pollen prior to storage improvesviability of the stored pollen. Typical fresh pollen moisture content isabout 60%. Moreover, dehydration of the pollen during a fieldconditioning step combined with a specific relative humidity levelduring storage may provide optimal pollen storage conditions to preventand/or reduce loss of viability. The equilibrium moisture content of thestored pollen may be optimized to maintain and/or slow loss ofviability. In some maize examples, equilibrium moisture content of about45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%,about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, and/orabout 58% may maintain and/or slow loss of viability. Moreover, strivingfor an optimized equilibrium moisture content has shown to providebetter results than simply storing pollen in a high humidityenvironment.

The addition of gases, such as nitrogen (N₂) or carbon dioxide (CO₂),serve several functions in preparing the pollen for storage, as shown inExamples 10, 11, and 12 below. The first function is that the gases canserve as drying agents. The moisture content of these gases is typicallybelow 1.0%. Constantly flowing a refreshed source of gas to the chamberwhere the pollen is being retained ensures that the moisture liberatedfrom the pollen is exhausted from the system. The gases also serve todisplace oxygen from the chamber. Oxygen is required by the pollen formetabolic activity, and promotes accumulation of reactive oxygen species(ROS). Reducing metabolic activity helps field condition the pollen andprepare it for storage.

The chamber containing the pollen intended for storage is subjected to apositive pressure, or a flow of treated air to the chamber. Thispressure may be established through the pumping of treated air into thechamber as demonstrated in Examples 10, 11, and 12. The positivepressure of the treated air flow may extract, add, or maintain pollenmoisture and further may extract, add, or maintain chamber moisture,allowing for the careful control of the relative humidity (RH) level.The RH and temperature of the air can be established using anycombination of desiccants, salt solutions, heat, or other treatments tomaximize its effectiveness in controlling drying of the pollen.

During the phase of drying the pollen to 15 to 35% moisture content, theair in the chamber containing the pollen is maintained from about −10°C. to about 10° C., including about −10° C., about −9° C., about −8° C.,about −7° C., about −6° C., about −5° C., about −4° C., about −3° C.,about −2° C., about −1° C., about 0° C., about 1° C., about 2° C., about3° C., about 4° C., about 5° C., about 6° C., about 7° C., about 8° C.,about 9° C. and about 10° C. This can be achieved by placing the chamberin a chilled room or by introducing chilled air into the chamber througha variety of means.

Optionally, during the period of pollen drying, the pollen containedwithin the chamber may be agitated mechanically. The agitation serves toexpose the maximum surface area of the pollen to the air in the chamber,thereby providing for more even drying and preventing clumping of thepollen. This may be achieved in a number of different ways, includingthe use of vibration, forced air, rotation, or other means.

Once the pollen has reached the optimal moisture content of 15 to 35%moisture, it can be stored. Storage can be achieved by placing thechamber of pollen in liquid nitrogen, in freezing conditions of −80° C.to 0° C., in refrigeration, or at atmospheric conditions. The choice ofconditions in which to store the pollen may depend upon the timeframe inwhich the pollen is expected to be used.

As explained in detail in the preceding paragraphs, maximizing andextending the viability of pollen using the disclosed method requirescareful manipulation of a number of factors within defined ranges.Laboratory-generated data using maize pollen as a test species haveindicated the conditions for improving pollen viability includemodification of one or more conditions within the chamber to fall withinthe disclosed ranges as set forth below.

The relative humidity of the chamber containing the pollen may bemaintained at any value ranging from about 75% humidity to about 100%humidity, including about 75%, about 76%, about 77%, about 78%, about79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%,about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%,about 99%, and about 100% humidity.

The temperature of the chamber containing the pollen may be maintainedfrom about −10° C. to about 10° C., including about −10° C., about −9°C., about −8° C., about −7° C., about −6° C., about −5° C., about −4°C., about −3° C., about −2° C., about −1° C., about 0° C., about 1° C.,about 2° C., about 3° C., about 4° C., about 5° C., about 6° C., about7° C., about 8° C., about 9° C. and about 10° C.

The concentration of oxygen (02 gas) present in the chamber may bemoderated from about the prevailing atmospheric percentage to about 0%,including about 21%, about 20%, about 19%, about 18%, about 17%, about16%, about 15%, about 14%, about 13%, about 12%, about 11%, about 10%,about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%,about 2%, about 1%, and about 0% oxygen.

The concentration of nitrogen (N₂ gas) present in the chamber may bemoderated from about atmospheric percentage to about 100%, includingabout 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%,about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about97%, about 98%, about 99%, and about 100% nitrogen.

Furthermore, the atmospheric pressure and carbon dioxide (CO₂ gas)concentration within the chamber may be modified to improve viability asoutlined below. The atmospheric pressure inside the chamber may bemodified in a manner that supports the tight control of humidity levels.Furthermore, the modification of air pressure allows for the preventionof oxidation of the pollen. Appropriate atmospheric pressures range fromabout 15 kPa to about 150kPa, including about 15 kPa, about 20 kPa,about 25 kPa, about 30 kPa, about 35 kPa, about 40 kPa, about 45 kPa,about 50 kPa, about 55 kPa, about 60 kPa, about 65 kPa, about 70 kPa,about 75 kPa, about 80 kPa, about 85 kPa, about 90 kPa, about 95 kPa,about 100 kPa, about 105 kPa, about 110 kPa, about 115 kPa, about 120kPa, about 125 kPa, about 130 kPa, about 135 kPa, about 140 kPa, about145 kPa, or about 150 kPa.

The concentration of CO₂ present in the chamber may be moderated fromabout atmospheric percentage (about 0.04%) to about 100%, includingabout 1%, about 10%, about 20%, about 30%, about 40%, about 50%, about60%, about 70%, about 75%, about 80%, about 81%, about 82%, about 83%,about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%,about 97%, about 98%, about 99%, and about 100% CO₂.

Following the modification of the conditions inside the chamber, thepollen is maintained in the defined conditions for a period of time toallow the improvements in pollen viability to take place, and as aresult, extending the viability of the pollen. Even such fieldconditioning for a short period of time, including but not limited to 60minutes, can improve the viability of the pollen and extend the durationof the viability window. Initial laboratory results have shown theoverall viability of maize pollen improved significantly, more thanfour-fold for many genetic backgrounds, after 24 hours in the fieldconditioning environment (see Example 3). In addition, the window ofviability of maize pollen has been extended with this field conditioningmethod. Laboratory results have shown that maize pollen, which normallyhas a viability window of approximately less than 2 hours, has aviability window extended to approximately 17 days, following treatmentwith the method of the invention (Example 8).

Accordingly, in one optimal embodiment, pollen may be collected fromactively shedding plants and placed into a preservation chamber. Thepreservation chamber may include a constant flow of nitrogen gas. Thepreservation chamber is adjusted for a target pollen moisture content,such as about 30%. As the pollen moisture content decreases, thetemperature in the chamber can slowly be adjusted downward, also, suchas to about -5° C. Preferably the temperature adjustment occurs withoutfreezing the pollen. Similarly, the relative humidity levels in thechamber can also be adjusted to increase or decrease the rate of pollendehydration. The relative humidity in the chamber may eventually beincreased if necessary to stabilize the final pollen moisture content atabout 30%. Optimally, this process is accomplished in approximately 100minutes, although other time periods may be used without departing fromthe scope of the invention and with success.

Pollen subjected to such a method may be used in any application wherepollen is a commercial or experimental unit. In one example, the fieldconditioned and preserved pollen may be used to produce seed, hybrid,parent, or otherwise, in any setting, including but not limited to alaboratory, greenhouse, and field. In another example, the fieldconditioned and preserved pollen may be used to produce grain, hybrid orotherwise, in any setting, including but not limited to a laboratory,greenhouse, and field. Moreover, as discussed above, such a method maybe applied to pollen from the Poaeceae (Gramineae) family of plants, aswell as any other plant species wherein it is desired to field conditionand preserve pollen.

The field conditioning and preservation techniques disclosed in thisinvention are intended to successfully treat and preserve pollen suchthat the field-conditioned and/or preserved pollen maintains itsviability to the extent that about 4 to about 20 grains of pollen aresufficient to successfully pollinate an embryo.

As noted above, although the invention has applicability to maize andthe maize industry, the invention is applicable to storage andpreservation of all types of pollen. In another example, rice pollen maybe successfully preserved, as provided in Example 9 below. Specifically,in the provided Example, rice pollen was preserved at 4° C. and 100%relative humidity for 20 hours. The preserved pollen showed agermination rate of 25%, proving that the methods of the presentinvention are applicable to other plant species. Moreover, using theexperimental methods described herein, one skilled in the art mayoptimize the present invention for a desired species of pollen. Thefollowing examples illustrate the present invention in more detail andare illustrative of how the invention described herein could beimplemented in maize.

EXAMPLE 1 Effect of Genotype on Pollen Health

A study was undertaken to determine the effect of plant geneticbackground on the viability and overall health of freshly-shed pollen.Fresh pollen was collected from field grown maize plants of eightgenetic backgrounds in Grimes, Iowa at approximately 11:30 am on Aug.14, 2016. The genetic backgrounds were selected to represent a broadsample of heterotic groups known in maize breeding. Tassels of theplants were vigorously brushed free of adhering anthers and pollen at9:00 am the same day. Pollen from tassels of approximately 10 plants ofeach genetic background was collected into a separate paper bag. Pollenwas separated from debris by passage through a screen (150 micron poresize) and immediately placed into field conditioning at 4° C. for 80minutes. Field conditioning involved spreading a thin layer of pollen inthe bottom of a 15 cm petri plate that had prechilled water-moistenedblotter paper held in the upper half of the plate. Following fieldconditioning, pollen within each plate was gently mixed to obtainhomogeneity and a small portion assayed for viability. In vitrogermination was used to assess viability by incubating pollen in anartificial media (438 mM sucrose, 1.6 mM H₃BO₃, 3.0 mM CaCl₂-H₂O) forone hour at 22° C. Duplicate assays were conducted. With the aid of amicroscope, percent germination was measured as the number of pollengrains with a pollen tube length more than twice the diameter of thegrain from a random sample of typically 200 grains.

Many factors can affect the viability of pollen, including geneticbackground. As demonstrated in Table 1, the viability of pollen amongeight diverse genetic backgrounds of maize differed significantly byapproximately two-fold when sampled on a single day. Genetic lines ofmaize are reported to show large differences in the tolerance of pollento stress, particularly heat stress and vapor pressure deficits thatlead to dehydration. For these reasons, when assessing methods forpreservation of pollen, it is important to segregate variation inviability due to genetic differences from that which can be caused byother factors, such as handling procedures and storage conditions.Equally important, it is prudent to evaluate more than a single geneticbackground when determining best practices for storing pollen.

TABLE 1 Difference in viability of pollen among eight diverse geneticbackgrounds of maize In Vitro Percent Genetic Germination Background(Mean +/− SE) AATH3 54 (+/−1) 207 56 (+/−0) H99 28 (+/−0) CM105 63(+/−7) C103 45 (+/−8) OQ101 63 (+/−9) LH162 50 (+/−0) OH43 39 (+/−4)

EXAMPLE 2 Effect of Laboratory Conditions on Pollen Health

A study was undertaken to determine the effect of ambient laboratoryconditions on the viability of freshly-shed pollen. Fresh pollen wascollected from tassels of plants grown in the greenhouse and exposed toambient environmental conditions in the laboratory. Pollen was takenfrom two unrelated genetic backgrounds of maize, Yukon Chief and SilverChoice, to preclude drawing conclusions based on a sole geneticmaterial. Immediately after collection, pollen was separated from debrisby passage through a screen (150 micron pore size) and placed in a thinlayer on a paper sheet set on the laboratory bench. The pollen was leftin this condition without any special precautions to control thelaboratory ambient temperature and relative humidity, which were 21-23°C. and 30-34%, respectively, over the course of the experiment. Thepollen was mixed to obtain homogeneity and sampled for measurement ofviability at 0, 1, 2, 3 and 23 hours after the start of the experiment.In vitro measured as described in Example 1, using using a random sampleof about 100 grains per measurement. Results are presented in FIG. 1.

The percent germination of Yukon Chief declined rapidly with exposure tolaboratory ambient conditions, beginning at 88% and declining to 4%within three hours. After 23 hours of exposure only a trace ofgermination could be detected among grains in the pollen. Pollen ofSilver Choice followed a similar pattern except the time zero value waslower than the viability at one hour exposure. The time zero value isbelieved to be an outlier.

The environmental conditions of the laboratory were very unfavorable formaintenance of viability in pollen of the two genetic backgroundsexamined. The occurrence of high temperature stress in maize pollen isnormally known to occur at temperatures in excess of 35° C. and, sincethe laboratory was 21-23° C., it is most likely that viability was lostquickly from these samples because the ambient relative humidity rapidlydehydrated the pollen over a three-hour period. Dehydration is known toaffect the viability, and sometimes, fertility, of maize pollen.

EXAMPLE 3 Pollen Field Conditioning: Cold and High Relative Humidity

A study was undertaken to determine whether the viability of field-shedpollen could be improved to reverse the negative effects of dehydrationcaused by vapor pressure deficit stress. Fresh pollen was collected fromfield grown maize plants of eight genetic backgrounds in Grimes, Iowa atapproximately 1:45 pm on Aug. 10, 2016. The ambient temperature andrelative humidity were 31.7° C. and 72%, respectively, duringcollection. The genetic backgrounds were selected to represent diverseheterotic groups known in maize breeding, and included hybrid and inbredlines. The time of collection, mid-afternoon, was chosen intentionallybecause the vapor pressure deficit is typically more unfavorable topollen health when temperatures are elevated and relative humidityreduced, compared to times earlier in the day. Tassels of the plantswere vigorously brushed free of loosely held anthers and pollen at 9:00am the same day and each subsequently covered with a paper bag. At leastsix plants of each genetic background were bagged. Upon collection,tassels were shaken in the bags and material retrieved from the bags ofeach genotype passed through a screen (150 micron pore size) to collectthe pollen. Each genetic pool of pollen was mixed thoroughly to obtainhomogeneity. Assays for in vitro germination were immediately initiatedin the field by sampling the pollen collected for each background (assaydescribed in Example 1). In addition, each pool was sampled to measurepollen moisture content (PMC), which was calculated after weighing50-100 mg pollen before and after drying at 104° C. for 24 hours.Promptly, the pools of pollen were subjected to field conditioning, heldthere for 120 minutes, and assayed again for in vitro germination, asdescribed in Example 1. A single assay was conducted for each geneticbackground at each time and approximately 335 pollen grains wereexamined for each test.

Fresh maize pollen typically has a PMC of roughly 60%, but in thisexperiment PMC values of freshly collected pollen ranged from22.8-46.9%, with an average of 35.4% (FIG. 2). The ambient conditions atthe time of pollen collection, 31.7° C. and 72% relative humidity,resulted in a vapor pressure deficit that caused the pollen that wasloosely bound in the tassels to dehydrate. Genetic differences in PMC offreshly collected pollen were apparent, as similarly reported by otherresearchers.

Pollen from each genetic background was tested for in vitro germinationimmediately after collection and again two hours later following fieldconditioning at approximately 4° C. and 100% relative humidity. FIG. 3shows that at time-0, prior to field conditioning, pollen germinationwas less than 10% for all genotypes but pollen viability was increased,more than four-fold for many genetic backgrounds, following two hours ofincubation at high humidity and low temperature. Viability of thedehydrated pollen was revived more for some genetic backgrounds thanothers but, as a rule, field conditioning the stressed pollen improvedits ability to germinate and form pollen tubes that are required inorder for pollen to fertilize ovules and form seed. Reversing thenegative effects of stress and dehydration on pollen viability andfertility through field conditioning can play an important role instoring pollen in preparation for its use as a pollination supplement toimprove the purity and yield of seed produced in seed and grainproduction.

EXAMPLE 4 Pollen Field Conditioning: Effect of Temperature VersusRelative Humidity

A study was undertaken to determine whether the chilling or the highrelative humidity produced the rescue effect noted in Example 3, and todetermine how quickly this effect occurred. Fresh pollen was collectedfrom field grown maize plants of four genetic backgrounds (Mo17, C103,AATH1, AATH3) in Slater, Iowa on Jul. 27, 2016. The genetic backgroundswere selected to represent diverse maize germplasm, and included hybridand inbred lines. Tassels were vigorously brushed free of loosely heldpollen, bagged, and pollen collected as described in Example 3. Assaysfor in vitro germination were immediately initiated in the field andpollen sampled for PMC as also described in Example 3. Promptly uponcollection, the pools of pollen were placed into field conditioning,using thin layers of pollen in hydrated 6 cm petri plates, as describedin Example 1. The samples were field conditioned, at the field location,at either 4° C. or 22° C. for two hours. Pollen was sampled every hourfor in vitro germination, as detailed in Example 1. A single assay wasconducted for each genetic background at each time point andapproximately 206 pollen grains were examined for each test.

Pollen field conditioning was performed with a single, high level ofrelative humidity (ca. 100%) and two incubation temperatures. Afactorial treatment scheme with level of relative humidity for fieldconditioning was not possible at the test location. Hence, theexperiment examined how field conditioning with high humidity influencedpollen viability compared to its initial level (i.e., time of pollencollection) and whether the combination of high humidity with lowtemperature provided any unique benefit.

The PMC of pollen at time of collection ranged from 51.9% to 59.2%, sonone of the inbreds or hybrids demonstrated severe dehydration or stressof the pollen. Average viability of the four genetic backgrounds acrosstreatments is shown in FIG. 4. Pollen hydration was good, to excellent,at the start of field conditioning but viability was slightly low, asindicated by pollen average in vitro germination rates of 39%. After 60minutes or less, at “normal” temperature (22° C.), hydration of freshpollen increased germination by 21%, on average. Hydration plus coldtreatment (4° C.) increased germination 33%, on average. In summary,field conditioning the pollen elevated low viability samples within onehour, primarily due to the presence of high humidity, but additionalbenefit was gained from storage at low temperature.

Details of the effect of field conditioning on revitalization of pollenviability is shown for each genetic background in FIG. 5. The importanceof examining multiple genotypes in order to gain a clearer picture ofhow field conditioning and storage processes affect maize, in general,are again evident. Genotypic differences occurred for, (a)viability attime of pollen collection (e.g., C103 vs. AATH1), (b) revitalizationresponse to high humidity (e.g., Mo17 vs. C103) and (c) advantageobtained by field conditioning at low temperature (e.g., Mo17 vs. C103).Despite genetic differences, it was nearly always true that theviability of fresh pollen can be improved by short term storage at highhumidity and low temperature.

EXAMPLE 5 Pollen Field Conditioning: Vacuum Treatment

Experiments conducted had shown an ability to collect pollen underenvironmentally stressful conditions and to improve its viability byfield conditioning it under specific temperature and relative humidityconditions. It was theorized that oxidative stress could be responsiblefor deterioration of germinability when pollen is stored over a periodof days. Experiments were conducted to remove oxygen from the storageenvironment to determine whether this improved the duration of viablepollen storage. Anoxic conditions were obtained by vacuum evacuation.

Fresh pollen was collected from field grown maize plants of nine geneticbackgrounds (B14, C103, SQ, B37, 207, LH162, AATH1, AATH3) between 9:45am and 1:10 pm in Slater, Iowa on Jul. 22, 2016. The genetic backgroundswere selected to represent diverse heterotic groups known in maizebreeding, and included hybrid and inbred lines. Pollen of hybrid AATH1was sampled from plants grown in two different areas of the field.Tassels were vigorously brushed free of loosely held pollen and baggedthe evening before the day of use. Pollen was collected as described inExample 3.

Each genetically unique pool of pollen was sampled for PMC immediatelyafter collection. PMC was measured as described in Example 3. Each poolof pollen was then split into halves and promptly placed as a thin layerin the lower half of a 6 cm petri plate. Each plate was loosely coveredwith a paper fiber wipe (Kimwipe). As collected, covered petri plateswere placed on a rack held in a 3.78 L stainless steel vacuum chamber.In the bottom of each chamber was 200 mL of prechilled water. Each tankwas covered with an acrylic lid tightly secured by rubber bands orvacuum. One half of each pollen sample was placed in a chamber withatmospheric pressure (101.3 kPa) or a vacuum (50.5 kPa pressure). Thechambers were surrounded by ice in coolers while samples were collectedin the field and then transferred to a 5° C. incubator in thelaboratory. After six days of storage, chambers were opened, pollensamples mixed to homogeneity, sampled for PMC, and pollen assayed for invitro germination as described in Example 1. A single assay wasconducted for each genetic background and an average 298 pollen grainswere examined for each test.

Pollen became more dehydrated as collection occurred later in the day(FIG. 6). This response has been observed often in our experiments andby others (Kaefer, K. A. C., et al. (2016) Afr J Agr Res, 11(12),pp.1040-104). When stored for six days at high humidity, most samplesfailed to hydrate to a great degree, especially those severelydehydrated at time-0 (compare FIGS. 6 and 7). Therefore, pollen of somegenetic backgrounds (e.g., B37, AATH1, AATH3) was stressed before andduring storage and failed to germinate, regardless of storage pressure.For other backgrounds, the PMC ranged from 45-55% following storage andpollen remained viable for six days (FIGS. 7, 8). Storage at the lowerpressure caused pollen, on average, to become slightly drier. There werewide differences in the effect of pressure on PMC depending upon thegenetic background and initial PMC. Among samples viable after six daysstorage, pollen germination was slightly greater when stored at reducedpressure and oxygen level (FIG. 8). Again, treatment effect differedacross genetic backgrounds but the data suggested that viability duringstorage with high humidity and low temperature is generally improved ifstored below atmospheric pressure.

EXAMPLE 6 Pollen Field Conditioning: Vacuum Titration

Given the recognized advantage of storing pollen under vacuum,experiments were designed to titrate the level of vacuum necessary formaximizing the preservation of the pollen's viability.

Fresh pollen was collected from field grown maize plants of six geneticbackgrounds (OH43, Mo17, C103, H99, LH162, OQ101) between 10:30 am and1:10 pm in Slater, Iowa on Jul. 20, 2016. The backgrounds representedmaize inbreds of diverse heterotic groups. Tassels were vigorouslybrushed free of loosely held pollen and bagged the evening before theday of use. Pollen was collected and immediately (i.e., Time-0) sampledfor determination of PMC and in vitro germination, as described inExample 3. Each genetic pool of pollen was then split into sixequally-sized fractions. Each fraction was placed as a thin layer in thebottom of a 6 cm petri plate that was subsequently covered with a paperfiber wipe. One plate of each genetic background was set on a rack thatwas placed into one of seven 3.78 L stainless steel vacuum chambers. Inthe bottom of each chamber was 200 mL of prechilled water. Each chamberwas covered with an acrylic lid and held at ambient pressure (101.3 kPa)and 5° C. for 24 hours. After one day of field conditioning, one of thechambers were briefly opened and pollen sub sampled again for in vitrogermination assay. Air was then evacuated from the chambers to 84.3,67.4, 50.5, 33.5, 16.6, or 8.5 kPa pressure. One chamber remained at101.3 kPa. All chambers were then incubated at 5° C. for six days beforea final measurement of PMC and in vitro germination. A singlegermination assay was conducted for each genetic background at each timeand approximately 285 pollen grains were examined for each test.

At Time-0, when pollen was initially collected in the field, most pollensamples were fairly well hydrated, as indicated by an average PCM of49.8% (FIG. 9). Only pollen of inbred OQ101 was dehydrated, with a PMCof 34.1%. Despite these levels of hydration, viability of all pollenbackgrounds was poor and average in vitro germination was only 7.4%(FIG. 10). One day later, after field conditioning in a high humidity,low temperature environment, viability was greatly improved and in vitrogermination averaged 42.4%. This response was very similar to thatdescribed in Example 3 (FIG. 3). Following revitalization provided by 24hours of field conditioning, the samples were ready to be furtherincubated at high humidity and low temperature, but now under reducedlevels of pressure and oxygen.

Storage for six days at high humidity and low temperature did not changethe average PMC of pollen maintained at atmospheric pressure (101.3 kPa)or 84.3 kPa, But as storage pressure and oxygen level declined furtherwith increasing vacuum, pollen became more dehydrated. Average PMCfollowing six days of storage declined from 50.1% at atmosphericpressure to 37.1% at pressures of 33.5 kPa, or lower. Patterns among thegenetic backgrounds were all similar, in that PMC declined as storagepressure declined.

Two inbreds, H99 and OQ101, did not maintain viability under any vacuumtreatment after six days of storage (FIG. 10). Inbred C103 tended todecline in viability as pressure and oxygen were reduced, very similarto that observed for C103 in Example 5 (FIG. 8). The other three inbredstested in this example displayed a pattern where viability duringstorage was improved if pollen was stored under a slight vacuum withpressures of 67-84 kPa (FIG. 10). On average, the best level of vacuumfor storage was 84.3 kPa, where viability was nearly doubled compared tostorage at atmospheric pressure. Genotypes differed in how well storageunder a slightly reduced pressure improved longevity of the pollen but,as a rule, maize pollen can maintain its viability better if storedunder a slight vacuum with high humidity and low temperature. This isthe first report to demonstrate the advantage of, and optimization for,preserving Gramineae pollen viability at reduced pressure and/or reducedoxygen.

EXAMPLE 7 Pollen Field Conditioning: Vacuum Time Course Study

Experiments were designed to improve understanding of the dynamics ofvacuum-enhanced storage by developing a time course study to examine theeffect of timing on the preservation of the pollen's viability.

Pollen was collected from field grown plants of eight geneticallydiverse maize backgrounds, as before (Example 1). After the pollen wasfield conditioned with high humidity and low temperature for 80 minutes,the rate of in vitro germination and PMC were measured (refer toExamples 1 and 3 for method of measurement). Each pool of pollen wasthen divided into two, and each half was spread as a thin layer in a 6cm petri plate covered with a paper fiber wipe. The plates were placedon a rack in one of two 3.78L stainless steel vacuum chambers. Withpollen of each genotype occurring in each chamber, acrylic lids weresecurely attached. One chamber remained at atmospheric pressure (101.3kPa) and from the other, air was withdrawn to establish a pressure of67.4 kPa. The chambers were incubated at 5° C. and very briefly openedafter 3, 5, and 8 days of storage to again sample pollen pools for invitro germination. Measurements of in vitro germination were conductedin duplicate on Day-0 of storage but as single assays at all othertimes. An average of 254 pollen grains were examined for each in vitrogermination assay.

After collecting pollen from tassels and field conditioning it for 80minutes, the PMC ranged from 50.0 to 60.8% across the geneticbackgrounds. Hence, the hydration state of this freshly-shed pollen wasgood. Viability of these pollen samples on Day-0 is listed in Table 1.Five of the eight backgrounds displayed values of 50%, or greater.

In general, the viability of pollen declined during the eight days ofstorage (FIG. 11). Deterioration of viability occurred more quickly forsome genetic backgrounds (e.g., background “207”) than others but,overall, storage under vacuum (67.4 kPa) allowed pollen to maintain ahigher viability for each period of preservation. Table 2 shows that,averaged across the eight genetic backgrounds, even after only threedays of storage viability was 48% better when pollen was held at reducedpressure. By eight days of storage the advantage of vacuum preservationwas 82%, compared to storage at atmospheric pressure. This againdemonstrates that the longevity of maize pollen viability, andtherefore, fertility, is extended with preservation at reduced pressureand/or oxygen level.

TABLE 2 Average viability of pollen from eight genetic backgroundsstored at atmospheric pressure (101.3 kPa) or under slight vacuum (67.5kPa). Percent In Vitro Improvement Germination (%) with Storage StorageDays of Storage at at at Storage 101.3 kPa 67.5 kPa 67.5 kPa 3 23.3 34.548.4 5 13.0 19.1 47.1 8 5.2 9.5 82.5

Storage of maize pollen is proven to be better under vacuum. Viabilityof maize pollen, a member of the Gramineae family, remains at a higherlevel with vacuum storage compared to storage at atmospheric pressureand the mechanism is quickly functional, within three days, or less. Itis best to combine preservation of Gramineae pollen under vacuum withstorage at high humidity and low temperature, two other environmentalconditions proven to favor pollen viability and reduce its deteriorationwhen held at non-freezing temperatures. Because viability remains higherwith maize pollen stored under vacuum, it can be expected thatvacuum-stored pollen can be preserved for longer periods of time beforeloss of viability occurs. Not all levels of vacuum evacuation facilitateextension of pollen viability during storage and, in the case of maize,optimum conditions are approximately 67-84 kPa of pressure. Furthermore,not all genetic forms of maize pollen respond in an identical fashion tovacuum storage and some genetic backgrounds display more, or less,benefit from handling in this manner. Our testing with a wide diversityof maize germplasm and the like favorable response to vacuum storageacross the backgrounds, however, indicate that it is reasonable toassume that most, if not all, forms of maize will display improvedstorage of pollen when stored under pressure of approximately 67-94 kPa.This practice further enables development of commercial services wherelarge quantities of pollen are collected at an initial time, held incontrolled environmental conditions for extended periods, and used at alater time to provide supplemental pollination to seed or grain cropsfor the purpose of improving yield and/or purity.

EXAMPLE 8 Pollen Field Conditioning: Dehydration Before Storage

This example outlines experiments conducted with dehydration of pollenbefore storage.

Freshly-shed maize pollen typically has a PMC of about 60% (Fonseca andWestgate (2005) Field Crops Research 94: 114-125). The PMC may be lowerand the pollen partially dehydrated if the pollen is shed during periodsof stress, such as when unfavorable vapor pressure deficit exists(Example 3). Stress commonly compromises pollen viability. In thesecircumstances, the state of pollen hydration can often be improved andviability revived, either partially or in whole, through fieldconditioning (Example 4, FIG. 5). But full restoration of PMC andviability to “normal” levels is not always achieved and so it was ofinterest to investigate how dehydration prior to storage affected pollenviability.

Pollen used in these experiments was sourced from tassels taken fromfield or greenhouse grown maize. The tassels were detached from plantsand transported to the laboratory where 4 cm of each tassel's stem wascut off and discarded. The freshly cut ends of tassels were insertedinto a beaker containing water. The tassels were kept in an incubatorprogrammed for 25°/15° C. day/night temperature and 65%/80% day/nightrelative humidity, as well as daytime lighting. Tassels were acclimatedto the incubator environment for at least 24 hours before collection ofpollen. Following acclimation, pollen was shaken from freshly-sheddingtassels approximately two to four hours after the start of the daytimeenvironmental conditions. The tassels were from a mix of several maizeinbred genotypes and one variety of sweetcorn. Although the geneticidentity of plants used was known in most cases, no attempt was made todetermine the proportional contribution each genotype made in providinga pool of pollen for daily use. Collected pollen was separated fromdebris by screening (150 micron pore size), immediately placed intofield conditioning (Example 1), and held as such for 2-24 hours.

In experiment-A, field conditioned pollen was dehydrated to varyingdegrees by passing dry nitrogen gas over the pollen at 9° C. The pollenwas held on a screen (45 micron pore size) in a small (1.0 L) desiccatorand nitrogen continuously streamed through the desiccator in a mannerakin to that used by others (Barnabas, B. and Rajki, E. (1981). Ann Bot,48(6), pp.861-864). Subsamples of pollen were taken from the desiccatorat 30 minute intervals, analyzed for viability, and loaded into uncapped0.5 mL polyethylene microfuge tubes until the tube was approximately 20%full. The tubes were placed in a 3.78 L stainless steel sealed chamberthat had a pressure of 67.4 kPa (i.e., vacuum) and was stored at 5° C.The chamber was removed after 6, 9, and 17 days of storage to retestviability.

In experiment-B, field conditioned pollen was measured for PMC andviability before being treated with varying conditions of relativehumidity. Pollen was spread as a thin layer in aluminum weigh boats (4.3cm dia.) which were placed on a raised platform in 0.5 L glass storagecontainers that seal air-tight. The glass containers containedapproximately 200 mL of water or a saturated solution of (ACS reagentgrade) potassium sulfate, potassium nitrate, or strontium nitrate which,respectively, produce a calculated relative humidity of 100%, 98.5%,96.5% or 92.4% in an enclosed container at 55° C. (Greenspan, L., (1977)J Res Nat Bur Stand, 81(1), pp.89-96). The glass containers were storedat 5° C. and opened for less than 20 seconds after 6 and 11 days ofincubation to subsample pollen and retest PMC and viability.

Viability in experiments A and B was measured by Impedance FlowCytometry (IFC) on an Amphasys AG (Lucerne, Switzerland) AmphZ30instrument. This device singulates pollen grains as they flow through amicrotluidic chip supplied with microelectrodes. Changes in theelectrical impedance (resistance) of the fluidic medium are measuredwhen cells pass through the applied electric field and discrimination ofdead versus live pollen cells is achieved by changes of the phase angleof the detected impedance signal (Heidmann I., Schade-Kampmann, G.,Lambalk, J., Ottiger, M. and Di Berardino, M., 2016. Impedance FlowCytometry: A Novel Technique in Pollen Analysis. PloS one, 11(11),p.e0165531). Roughly 3,000 pollen grains were measured for each assay.For determination of PMC, samples were handled as described in Example3.

Dehydration of pollen with nitrogen gas in experiment-A caused a steadydecline of PMC over a four hour period (FIG. 12). The PMC was 59.2% atthe start of dehydration treatment and 13.1% when treatment ended. Bymidway through dehydration with nitrogen, the PMC had declined to 45.6%.

A subsample of pollen, at each step of dehydration, was placed into lowtemperature, low pressure storage in a sealed chamber and held there for17 days. Since no attempt was made to control the level of water vaporin the chamber during storage, it is possible that the subsamples ofpollen further dehydrated, or hydrated, during storage. Relativehumidity in the sealed chamber was primarily determined by laboratoryambient air conditions at the time the chamber was closed. Laboratoryair typically had a relative humidity of about 30% at the time of yearexperiment-A was conducted.

The rate that pollen subsamples within the storage chamber ofexperiment-A could have further dehydrated, or hydrated, during storagewould also have been affected by the manner of pollen containment withinthe chamber. The subsamples were stored in the lower portion of aplastic conical tube, so the entire surface area of the pollen was notequally exposed to water vapor in the chamber, like what would haveoccurred if the samples were spread in a thin stratum.

In experiment-A, the viability of pollen declined over the 17-daystorage period, regardless of the initial PMC (FIG. 12). But it was verysurprising to see that viability of pollen subsamples, which weredehydrated to 50-55% PMC ahead of storage, deteriorated slower thanpollen not dehydrated before storage or pollen dehydrated to PMC levelsless than roughly 45%. In fact, pollen only slightly dehydrated beforestorage, from a PMC of 59.2% for field conditioned pollen to 55.0% after30 minutes dehydration treatment, still had a 19.1%-Live viability after17 days of preservation.

Pollen hydration level will normally come to “equilibrium moisturecontent” (EMC), which is controlled by temperature and relative humidityof the surroundings (Connor, K. F. and Towill, L. E. (1993) Euphytica,68(1), pp.77-84). Since the rate of viability loss of stored maizepollen was slower in experiment-A than typical, it was reasonable tohypothesize that the stored pollen did not reach EMC with the dry air(30% RH) in the chamber. Perhaps this was because of the pollen'splacement in a plastic, conical tube. The notion that only slightdehydration of fresh pollen is needed for best practice of preservingviability of the material was tested in experiment-B.

Maize pollen in experiment-B was stored with the intent of having thePMC at EMC reach approximately 45-55%. This approximates the PMC levelsof pollen that remained most viable during storage in experiment-A.Controlled relative humidity levels employed in storage chambers ofexperiment-B were chosen carefully and employed saturated solutions ofpotassium sulfate, potassium nitrate, and strontium nitrate (as well aspure water). Hence, the range of relative humidity used as treatment was92.4-100%. In the past, over more than 100 years, researchers havestored fresh maize pollen at varying levels of humidity and assessed thevalue of such treatment in extending viability (i.e., storability)during preservation (Andronescu, Demetrius I., The physiology of thepollen of Zea mays with special regard to vitality. Thesis for degree ofPh.D. University of Illinois. 1915); (Knowlton H. E., 1922. Studies inpollen with special reference to longevity. (Vol. 52). CornellUniversity); (Sartoris, G. B., (1942) Am J Bot, pp.395-400); (Jones, M.D. and Newell, L. C., (1948) J Amer Soc Agron 40:195-204). Their worktaught that storage at high humidity, typically evaluating 90 or 100%humidity, preserves viability of the pollen better than storage at lowerhumidity. But even with storage at 90 or 100% humidity, viability orfertility of the pollen was only maintained for a few, to 10, days andviability of the samples declined sharply over the period of testing.Never before has it been reported or known that storage and preservationof maize pollen at a relative humidity (e.g., 95-98%) that causes an EMCof about 45-55% is, in fact, a very unique condition of the pollen andone that provides maintenance of viability in storage undisputedlysuperior to that of storage which produces a greater or lesser PMC.

The PMC of pollen stored at varying relative humidity in experiment-Bdeclined in a strict, linear fashion as storage humidity declined (FIGS.13A and 13B). Little change in PMC occurred beyond six days of storage,meaning the EMC was reached, or nearly so, in that time. Storage atrelative humidity of 96.5 and 98.5% produced an equilibrium PMC (i.e.,EMC) of 43.8 and 53.5%, respectively, as was targeted. FIG. 14A and 14Bshows that maize pollen stored in conditions of relative humidity thatproduced an equilibrium PMC of 44 or 54% retained nearly its fullviability over an 11-day period of preservation. Pollen stored at 100%or 92.4% relative humidity lost 78 and 91% of its viability,respectively, after 11 days.

It is logical to believe that methods practiced and results obtained inexperiment-B provide an understanding of how to extend the viability ofmaize pollen, without freezing, for extended periods of time that wereheretofore unattainable. Even work reported by Nath and Anderson (Nath,J., & Anderson, J. O. (1975). Effect of freezing and freeze-drying onthe viability and storage of Lilium longiflorum L. and Zea mays L.pollen. Cryobiology, 12(1), 81--88) failed to prevent deterioration ofviability in unfrozen pollen and it's not entirely clear that theirobservations were based on in vitro pollen germination, as opposed topseudo-germination (Andronescu, Demetrius I., The physiology of thepollen of Zea mays with special regard to vitality. Thesis for degree ofPh.D. University of Illinois. 1915). An ability to maintain theviability of unfrozen pollen in a fashion analogous to that demonstratedin Example 8 further enables practices that aim to preserve Gramineaepollen so that it can be used for pollination and ovule fertilization ina temporal manner precluded by normal periods of receptivity of femaleinflorescences in crop plants. It can also provide flexibility in thepractice of cryopreservation and enhance the quality of pollen intendedfor such use.

EXAMPLE 9 Rice Pollen Preservation

In order to validate that the preservation protocol developed for maizeis compatible with the pollen from other plant species, testing wasconducted using rice as a pollen source.

For the experiment, pollen from 2 different rice genotypes, one fromAsia and the other from the southern United States, were collected fromactively shedding rice plants and bulked together. Pollen from the bulkwas aliquoted into 2 different types of destination vessels. In thefirst vessel type, a light dusting of pollen was applied across thebottom of two VWR brand 100 mm wide by 15 mm deep petri dishes. The lidsto the petri dishes were intentionally left off to allow the pollen tointeract with the pollen preservation environment. In the second vesseltype, just enough rice pollen was added to a 0.5 mL flip cap tube tofill the rounded area at the bottom of the tube. This pollen volume wasestimated at 5 μL in each tube. The lids to the tubes were closed afteradding the pollen. With the lids in the closed position, the temperaturein the tube could be changed, but the relative humidity could not bechanged.

Each pollen preservation vessel was placed into an 887 mL Rubbermaidstorage container with a sealable lid. Enough H₂O was added to fill thebottom of each Rubbermaid container prior to sealing the container.

Each sealed Rubbermaid container was placed into a 4° C. environment fora preservation period of 20 hours. The pollen preservation vesselsreceived one of two treatments. In treatment 1, the petri dishes weretreated at 4° C. with 100% relative humidity for 20 hours. In treatment2, the 0.5 mL flip cap tubes were treated at 4° C. for 20 hours. After20 hours of storage, each sample of pollen was placed in a germinationmedia which allows the pollen tube to grow if the pollen is viable.After 60 minutes of time in the media, images were captured. Each imagewas scored for number of pollen tubes relative to the overall number ofpollen grains present.

Results: Rice pollen stored in the petri dishes showed an overallgermination rate of 25% and 1% respectively after 20 hours of storage at4C and 100% relative humidity. The inventors have speculated that theseal on the Rubbermaid vessel which yielded 1% germination was notproperly sealed. Failure to seal properly would result in the actualhumidity in the vessel to fall well below the 100% total.

Rice pollen stored in the flip cap tubes showed an overall germinationrate of less than 1% after 20 hours of storage at 4C, and no control ofrelative humidity. It should be noted that this germination percent isconsistent with the petri dish pollen stored at 4° C. and 100% relativehumidity germinating in culture after 20 hours of preservation thatyielded 1% germination as both scenarios would reflect an environmentthat fail to keep humidity near 100%. FIG. 15 provides an image of thegerminated rice from the petri dish having a germination rate of 25%,while FIG. 16 provides an image of rice pollen stored at 4° C. withoutrelative humidity control.

EXAMPLE 10 Maize Pollen Preservation Using 75% Humidity and Positive AirFlow

This example outlines experiments conducted to prepare pollen forpreservation. Pollen used in these experiments was sourced from tasselstaken from field and greenhouse grown maize. The tassels were detachedfrom plants and transported to the laboratory where 4 cm of eachtassel's stem was cut off and discarded. The freshly cut ends of tasselswere inserted into a beaker containing water. The tassels were kept inan incubator programmed for 25°/15° C. day/night temperature and 65%/80%day/night relative humidity, as well as daytime lighting. Tassels wereacclimated to the incubator environment for at least 24 hours beforecollection of pollen. Following acclimation, pollen was shaken fromfreshly-shedding tassels approximately two to four hours after the startof the daytime environmental conditions. Across all experiments, tasselswere from 21 different maize inbred and hybrid genotypes (total of 21genotypes). Collected pollen from each genotype was maintained andidentified by genotype as separate pollen samples. Each pollen samplewas separated from debris by screening (150 micron pore size).

After collection of pollen, each sample was immediately sub-sampled andmeasured (time 0) for viability using amphasys, which is discussed indetail above. Initial viability readings confirmed that all pollensamples had high initial viability of greater than 60%. The remainingpollen for each sample was then placed on a forced air drying apparatuswith approximately 75% humidity (enabled by a saturated NaCl solution)at 5° C. and dried for approximately 20 hours. The samples were storedat −80° C. for time periods ranging from 4 hours to 38 days andcounting. There was great variation in storage time due to the fact thatthere were a limited number of females available in the greenhouse touse for cross-pollinations with the stored pollen on any given day, andtherefore several of the samples had to wait in −80° C. conditions untila female was available.

Samples were removed from the −80° C. storage conditions, placed on dryice and transported to the greenhouse to make pollinations. Onlyproperly shoot-bagged ears were used as females to avoid any risk ofescapes. Two to three weeks after pollination, the ears were observed todetermine if kernels had formed, indicating that the pollen was viableafter receiving the treatment described above. Pollen from 18 of the 21genotypes tested resulted in kernels being formed, indicating that thepollen was still viable after storage at −80° C.

FIGS. 17A, 17B, and 17C provides examples of confirmed viability ofpreserved pollen stored at −80° C. Specifically, the photographs shownin FIGS. 17A, 17B, and 17C reflect the results of pollinations conductedwith preserved pollen following storage periods of 4 hours to 38 days.The presence of developing kernels was assessed two to three weeks afterpollination or at kernel maturity, as shown in Table 3 below.

TABLE 3 Presence of developing kernels for example 10 wherein genotypeindicates the source of pollen and female indicates the inbredpollinated by the preserved pollen. FIGURE 16A 16B 16C Days PollenStored 1 1 38 at −80° C. Female 78 H99 H99 Age of Kernels 21 69 19 (DAP)

EXAMPLE 11 Maize Pollen Preservation Using Nitrogen Gas and PositivePressure

This example outlines an experiment conducted to prepare pollen forpreservation. Pollen used in this experiment was sourced from tasselstaken from the field. The tassels from a single sweetcorn hybrid weredetached and transported to the laboratory where 4 cm of each tassel'sstem was cut off and discarded. The freshly cut ends of tassels wereinserted into a beaker containing water. The tassels were kept in anincubator programmed for 25°/15° C. day/night temperature and 65%/80%day/night relative humidity, as well as daytime lighting. Tassels wereacclimated to the incubator environment for at least 24 hours beforecollection of pollen. Following acclimation, pollen was shaken fromfreshly-shedding tassels approximately two to four hours after the startof the daytime environmental conditions. Pollen samples were separatedfrom debris by screening (150 micron pore size).

After collection of pollen, a sub-sample was measured (time 0) forviability using amphasys as described above, which confirmed that thepollen had high initial viability of greater than 95%. The remainingsample was then placed on a forced nitrogen drying apparatus at 5-10° C.(temperature varied). The nitrogen gas served the dual purpose ofdepleting the environment of oxygen while also decreasing the humidity.Relative humidity varied during drying from 40% at time 0 to 12% after110 minutes. Three sub-samples of pollen were removed at 80, 85, 90, 95,100, and 110 minutes. The first sub-sample was used to measure pollenmoisture content (PMC) of the sample. This PMC is reported in Table 4.The second sub-sample was used to measure pollen viability beforefreezing. The third sub-sample was stored at −80° C. for 120 minutes andused to measure pollen viability after freezing. Table 4 shows percentviability of the pollen used in this experiment. Results show that thereis a range of PMCs from 15 to 35in which pollen is more stable and canbe stored more effectively while minimizing the drop in viability.

TABLE 4 Percent viability for pollen dried to different percent moisturecontent (PMC). Time % % on Humidity viable viable % dryer reading beforeafter drop in Sample (min) (%) PMC(%) freeze freeze viability 201-1 0 4064.4 97.1 28.2 71.0 201-2 80 27 35.0 67.4 28.6 57.6 201-3 85 26 31.171.6 37.1 48.2 201-4 90 21 28.5 70.7 24.7 65.1 201-5 95 19 24.1 34.018.0 47.1 201-6 100 17 19.1 53.6 15.9 70.3 201-7 110 12 17.4 31.7 5.782.0

EXAMPLE 12 Maize Pollen Preserved Using Nitrogen Gas, Positive Pressure,and Adjustable Humidity And Temperature

Pollen that has been collected from actively shedding plants is placedinto the preservation chamber. A constant flow of nitrogen gas flows tothe chamber. The nitrogen gas serves the dual purpose of depleting theenvironment of oxygen, which is required for metabolism to occur, whilealso decreasing the humidity, which accordingly begins reducing thepollen moisture content to low levels, such as a target level of about30%. As the pollen moisture content decreases, the temperature in thechamber can slowly be adjusted down to well below 0° C. (−5° C. forexample) without freezing the pollen. Similarly, the relative humiditylevels in the chamber can also be adjusted to increase or decrease therate of pollen dehydration. Concomitantly, the humidity in the chambercan also be adjusted up to stabilize the final pollen moisture contentat about 30%. Using PV=nRT, a final humidity value can be calculated tohold the preserved pollen at an equilibrium pollen moisture content of30%. This process can be accomplished in approximately 100 minutes.

EXAMPLE 13 Maize Pollen Preserved in Liquid Nitrogen

To test the potential of using maize pollen that has been rapidly frozenas a source of preserved pollen, an experiment was conducted todetermine what percent of maize pollen survives after being flash frozenin liquid nitrogen. According to Nath and Anderson (Nath. J., &Anderson, J. O. (1975). Effect of freezing and freeze-drying on theviability and storage of Lilium longiflorum L. and Zea mays L. pollen.Cryobiology, 12(1), 81-88), “Rapid freezing of pollen at rates ofapproximately 200 degrees C./min maintains the highest degree of viablepollen in combination with rapid thawing rates of 218″C/min. Rapidcooling and slow rewarming resulted in a substantial loss of pollenviability. This might indicate that intracellular ice crystals formedduring rapid cooling perhaps grow into larger ice masses during slowrewarming or storage at temperatures above −50° C.”

For the experiment, maize pollen was collected from several differentactively shedding genotypes and bulked into a single source tube. Toachieve a rapid freeze of the pollen, 50 mL of liquid nitrogen waspoured into a 100 mL borosilicate beaker. The fresh pollen was thendropped into the liquid nitrogen to achieve the rapid freeze. The pollenremained in the liquid nitrogen until the entire 50 mL volume had boiledaway. The rapidly frozen pollen was immediately added to roomtemperature maize pollen germination media to achieve a rapid thaw rateof the pollen.

The results of the germination test were captured in images which weresubsequently scored for percent pollen tube growth compared to overallnumber of pollen grains. The vast majority of the pollen rapidlydegraded during the pollen tube germination assay. Significant leakageof the cellular contents was noted as a large amount of debris becameevident in the media during the germination process. The finalgermination rate was scored at 1/221 grains of pollen, or less than0.5%. FIG. 18 shows the final germination media results from theabove-described rapidly frozen and rapidly thawed maize pollen. Overallgermination was scored at less than 0.5%.

Accordingly, as provided in the above, the methods of the presentinvention provide a number of advantages which have heretofore beenlacking in the industry. The present invention provides large scalepollen preservation methods. The methods of the present inventionmaintain and increase pollen viability during collection, as well asduring preservation. Moreover, the methods of the present invention areapplicable to, but not limited to, two specific advantageous situations.First, the method is applicable to instances where pollen will be usedwithin 30 days of collection in support of the current growth cycle atthe time of collection. Second, the invention allows indefinite storageduration, which allows for pollen to be stored for years prior todelivery as desired to receptive female plants.

Furthermore, the majority of prior pollen preservation methods rely onfreezing and subsequent freeze drying of the pollen (including, but notlimited to, the Greaves, et al. and Nath and Anderson methods describedabove) to achieve dehydration of pollen to conditions wherein the pollenmay be stored. However, the lengthy time necessary to freeze dry thepollen reduces the amount of time wherein the pollen may be used forfield applications. Further, the methods of the prior art do not controlthe pollen moisture content, which in some embodiments may be importantto preserving the pollen. Failure to fall within the sensitive pollenmoisture content range which allows pollen to be preserved results insignificant loss of viability. Moreover, the pressure requirements ofthe present invention allow for increased scalability and portabilityover methods of the prior art, which require more extreme pressureconditions. Methods of the current invention provide for quickdehydration of pollen and also enables the pollen to be held at theoptimum pollen moisture content.

Although various representative embodiments of this invention have beendescribed above with a certain degree of particularity, those skilled inthe art could make numerous alterations to the disclosed embodimentswithout departing from the spirit or scope of the inventive subjectmatter set forth in the specification and claims. In some instances, inmethodologies directly or indirectly set forth herein, various steps andoperations are described in one possible order of operation, but thoseskilled in the art will recognize that steps and operations may berearranged, replaced, or eliminated without necessarily departing fromthe spirit and scope of the present invention. It is intended that allmatter contained in the above description or shown in the accompanyingdrawings shall be interpreted as illustrative only and not limiting.Changes in detail or structure may be made without departing from thespirit of the invention as defined in the appended claims.

Although the present invention has been described with reference to theembodiments outlined above, various alternatives, modifications,variations, improvements and/or substantial equivalents, whether knownor that are or may be presently foreseen, may become apparent to thosehaving at least ordinary skill in the art. Listing the steps of a methodin a certain order does not constitute any limitation on the order ofthe steps of the method. Accordingly, the embodiments of the inventionset forth above are intended to be illustrative, not limiting. Personsskilled in the art will recognize that changes may be made in form anddetail without departing from the spirit and scope of the invention.Therefore, the invention is intended to embrace all known or earlierdeveloped alternatives, modifications, variations, improvements, and/orsubstantial equivalents.

1. A method of grain production, comprising: a. growing a plurality ofdesignated female plants that include both female and male components;b. collecting fresh pollen from designated male plants from a differentgenetic background; c. subjecting said fresh pollen to fieldconditioning conditions including: i. relative humidity ranging fromabout 50% to about 100%; ii. ii. temperature ranging from about −10° C.to about 10° C.; and iii. iii. air pressure ranging from about 15 kPa toabout 150 kPa; resulting in field conditioned pollen having a pollenmoisture content of about 40% to about 58%; and d. intentionallycross-pollinating, on one or more days, said designated female plantswith said field conditioned pollen; e. growing the designated femaleplants to maturity; and f. harvesting the grain produced by saiddesignated female plants wherein the female components of saiddesignated female plants are not covered to prevent undesirablepollination.
 2. The method of claim 1 wherein said field conditionedpollen is subsequently preserved in pollen preservation conditionsincluding a temperature ranging from about −10° C. to about 10° C.,wherein the air pressure is capable of adjustment and the fieldconditioned pollen is dehydrated to achieve a pollen moisture content ofabout 15% to about 35%, and the temperature and the relative humidityare adjustable and maintain the pollen moisture content at about 15% toabout 35%;
 3. The method of claim 1 wherein said method results in oneor more of the following: a. Increased grain yield, and b. Modifiedgrain characteristics.
 4. The method of claim 1 wherein said designatedfemale plant is any one or more of: a corn plant, a soybean plant, awheat plant, a rice plant, a sunflower plant, a canola plant, a sorghumplant, a barley plant, or a pearl millet plant.
 5. The method of claim 1wherein the intentional application of designated male plant pollen isconducted by any one or more of: mechanical means, pneumatic means,positive pressure means, negative pressure means, manual means, orcombinations thereof.
 6. The method of claim 1 wherein said designatedmale plant pollen has been harvested from one or more of a field, agrowth chamber, a greenhouse, a glasshouse, a shade house, a hoop house,a vertical farming facility or a hydroponic facility.
 7. The method ofclaim 1 wherein said designated male plant pollen is applied on morethan one occasion to the same designated female plant.
 8. The method ofclaim 7 wherein said designated male plant pollen is applied at the timewhich the female plant first becomes receptive to said pollen.
 9. Themethod of claim 3 wherein the modified grain characteristics include oneor more of grain size, grain content, or grain composition.
 10. Themethod of claim 1 wherein said designated male plant pollen is obtainedfrom sources optimal for application based on environmental conditions.11. The method of claim 10 wherein said environmental conditions areabiotic conditions.
 12. The method of claim 11 wherein said abioticconditions include at least one of drought, humidity, temperature,nitrogen availability or nutrient availability.
 13. The method of claim10 wherein said environmental conditions are biotic conditions.
 14. Themethod of claim 13 wherein said biotic conditions include at least oneof insect pest pressure or disease pressure.
 15. The method of claim 1wherein said designated male plant pollen is obtained from sourcesoptimal for application based on plant performance data.
 16. The methodof claim 1 wherein the designated male plant pollen influences thecharacteristics of the grain harvested from the female plant, whereinsaid influence impacts one or more of: i. grain oil content; ii. grainstarch content; iii. grain protein content; iv. grain oil composition;v. grain starch composition; and vi. grain protein composition.
 17. Themethod of claim 1 in which said designated male plant pollen is obtainedfrom a single genetic source.
 18. The method of claim 1 in which saiddesignated male plant pollen is obtained from multiple genetic sourcesand is combined prior to field conditioning.
 19. The method of claim 1in which said designated male plant pollen is obtained from multiplegenetic sources and is combined prior to application.
 20. A method ofpreventing undesirable pollination in grain production, comprising: a.growing a designated female plant that includes female components thatreceive designated male plant pollen; and b. collecting fresh pollenfrom designated male plants from a different genetic background; c.subjecting said fresh pollen to field conditioning conditions including:i. relative humidity ranging from about 50% to about 100%; ii. ii.temperature ranging from about −10° C. to about 10° C.; and iii. iii.air pressure ranging from about 15 kPa to about 150 kPa; resulting infield conditioned pollen having a pollen moisture content of about 40%to about 58%; and d. intentionally pollinating said designated femaleplant on one or more days with designated male plant pollen prior tosaid female plant being subjected to undesirable pollen on said one ormore days; wherein the female components of said female plant are notisolated to prevent undesirable pollination.
 21. The method of claim 20wherein said field conditioned pollen is subsequently preserved inpollen preservation conditions including a temperature ranging fromabout −10° C. to about 10° C., wherein the air pressure is capable ofadjustment and the field conditioned pollen is dehydrated to achieve apollen moisture content of about 15% to about 35%, and the temperatureand the relative humidity are adjustable and maintain the pollenmoisture content at about 15% to about 35%.
 22. The method of claim 20wherein the step of intentionally pollinating said designated femaleparent plant occurs during the morning.
 23. The method of claim 20wherein the level of cross-pollination is modified compared to naturalpollination.